There is a need for simple diagnostic tests for common diseases that can be executed by untrained personnel. Simpler tests would allow for home or doctor's office testing when current procedures require the analysis to be done by an outside laboratory. Possible benefits of simpler tests are decreased turn-around time and a reduction in cost. Representative examples are home pregnancy and glucose testing.
A common format for these simpler tests is the immunostrip format. Usually this format contains a mobile phase consisting of the test solution and a labeled, analyte-specific antibody. The analyte binds to the antibody and passes through a capture zone which contains immobilized analyte or an analyte derivative which is immunologically reactive with the antibody. The capture zone removes excess labeled antibody as the bound labeled antibody migrates to a detection zone.
There are numerous analytes whose detection by such a diagnostic test could benefit the public. Accurate, stable and reproducible tests are highly desirable. By using the osteoporosis marker deoxypyridinoline, which is illustrative of analytes, it is the intent of this invention to describe polymer bound analytes that provide advantages when used to immobilize the analyte onto the immunostrip's capture zone.
Collagen is present in various forms in all tissue. It is now well accepted that collagen has the form of amino acid chains cross-linked by the pyridinium crosslinks pyridinoline (PYD) and deoxypyridinoline (DPD). The pyridinium crosslinks are formed from three hydroxylysine residues, two of which are from the terminal (non-helical) peptides of the collagen molecule that are enzymatically converted to aldehydes before reaction and a third hydroxylysine situated in the helical portion of a neighboring collagen molecule. There have been described techniques in the literature for the measurement of pyridinoline in urine by use of an enzyme labeled antibody specific to pyridinoline to form a pyridinoline enzyme labeled complex which can be detected by an enzyme-linked immunosorbant assay. While the analysis for PYD is useful as a means of screening for osteoporosis and rheumatoid arthritis, its presence in connective tissue, as well as in bone, can cause skewed results. Accordingly, immunoassays for deoxypyridinoline, which is only found in bone, have become preferred over those for PYD in the early detection of bone degradation.
Testing for DPD can be accomplished by contacting a fluid test sample, e.g. urine, with a labeled antibody specific for DPD. A particularly convenient method for DPD determination involves the use of a test strip of the type depicted in FIG. 1. Referring to FIG. 1, strip 10 having a labeled anti-DPD antibody complex (typically with gold sol as the labeling material) binds with DPD in the fluid test sample in application zone 12 of the strip 10. The labeled DPD antibody and DPD in the fluid test sample which is applied to the application zone 12 of the strip 10 form an immunocomplex which migrates due to capillary action through the capture zone of the strip 14 and the optional detection zone 16. In the capture zone 14, there is immobilized DPD which captures unbound, labeled anti-DPD. The signal generated by the label on the captured anti-DPD is measured, such as by means of a reflectance spectrophotometer, and correlated with the results of replicate strips used to assay fluid test samples containing known amounts of DPD. As in classical competitive immunoassays, the intensity of the signal generated in the capture zone will be inversely proportional to the concentration of the DPD in the fluid sample. Labeled anti-DPD, which is not captured in the capture zone 14 because it had combined with DPD in the fluid test sample, is captured in the detection zone 16 by anti-mouse IgG, specific for a different epitope on the anti-DPD antibody than the previously mentioned active binding site for DPD on the labeled anti-DPD, which is immobilized in this zone. By measuring the spectral response from the capture and detection zones, and analyzing this response using an appropriate algorithm, the accuracy of the assay can be increased.
Nitrocellulose, commonly used to bind proteins and poly(ethylene glycols), is a preferred material for use in preparing the type of test strip illustrated by FIG. 1. Polysulfones, nylons or other porous membranes capable of adsorbing macromolecules also provide suitable strip material. A common technique to immobilize an analyte onto nitrocellulose or other solid support is to covalently bind the analyte to a protein that irreversibly adsorbs onto the solid support. Applying the resulting conjugate to the solid support results in an irreversibly bound analyte.
To provide a quality product, sensitivity, stability and precision are highly desirable. The object of this invention is to provide conjugates which provide such sensitivity, stability and precision.